**Purpose**
To understand the basic principle of alkaline plasmid extraction and to master the technique for isolating plasmid DNA using this method.
**Experimental Principle**
Plasmids are small, extrachromosomal, double-stranded, covalently closed circular DNA molecules that can replicate independently within bacterial cells. The alkaline lysis method is widely used in molecular biology laboratories to isolate plasmid DNA from bacterial cultures. This technique exploits the differences in topology between plasmid DNA and chromosomal DNA.
During alkaline treatment (pH 12.5), the chromosomal DNA denatures and becomes linear, while the plasmid DNA, although partially denatured, remains covalently closed. When the pH is neutralized with potassium acetate, the chromosomal DNA forms large aggregates and precipitates, whereas the plasmid DNA renatures quickly and remains in solution. After centrifugation, the plasmid DNA is collected in the supernatant. Further purification steps, such as ethanol or isopropanol precipitation, help remove contaminants like proteins and cellular debris, resulting in a relatively pure plasmid DNA sample.
**Key Features of Plasmids**
- Multiple cloning site (MCS)
- Selection marker (e.g., antibiotic resistance, LacZ)
- Autonomous replication origin (ori)
**Types of Vectors**
- Plasmid vectors
- Phage vectors
- Yeast artificial chromosomes (YACs)
- Retroviral vectors
- Expression vectors
- pBC SK
- T-vector
**Materials and Reagents**
- **Instruments**: Incubator shaker, laminar flow hood, autoclave, high-speed centrifuge, micropipette
- **Bacterial Strains**: *E. coli* DH5α containing pBC SK or recombinant plasmids
- **Reagents**:
- LB liquid and solid medium
- Solution I: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)
- Solution II: 0.2 M NaOH, 1% SDS
- Solution III: 3 M KAc, 5 M acetic acid (pH 4.8)
- Ampicillin (100 mg/mL)
- RNase A (10 mg/mL in 10 mM Tris-HCl, 15 mM NaCl)
- Chloroform, ethanol, 70% ethanol
**Experimental Procedure**
1. Inoculate a single colony of *E. coli* DH5α into 50 mL LB broth (with 0.1 mg/mL ampicillin) and incubate overnight at 37°C with shaking.
2. Transfer the culture to a 1.5 mL Eppendorf tube, centrifuge at 10,000 rpm for 1 minute, discard the supernatant, and repeat twice. Resuspend the pellet in 200 µL of Solution I.
3. Add 300 µL of Solution II, mix gently, and incubate on ice for 5 minutes.
4. Add 300 µL of Solution III, mix gently, and incubate on ice for another 5 minutes.
5. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new tube (approx. 600 µL).
6. Add an equal volume of chloroform, mix gently, and centrifuge at 12,000 rpm for 10 minutes.
7. Add pre-cooled isopropanol (equal volume), incubate at -20°C for 20–30 minutes.
8. Centrifuge at 12,000 rpm for 15 minutes.
9. Wash the pellet twice with 500 µL of 70% ethanol, centrifuge at 12,000 rpm for 3 minutes each time.
10. Dry the pellet at room temperature or under vacuum.
11. Dissolve the DNA in 25 µL of sterile water with 1 µL of RNase A at 37°C.
**Using the Biospin Plasmid Mini Kit**
1. Add 1–1.5 mL of overnight culture to a 1.5 mL tube.
2. Centrifuge at 10,000 rpm for 30 seconds, discard the supernatant. Repeat if needed.
3. Resuspend the pellet in 250 µL of Resuspension Buffer.
4. Add 250 µL of Lysis Buffer, invert 4–6 times gently.
5. Add 350 µL of Neutralization Buffer, invert 4–6 times.
6. Centrifuge at 13,000 rpm for 10 minutes.
7. Transfer the supernatant to a Spin column, centrifuge at 6,000 rpm for 1 minute.
8. Add 650 µL of Wash Buffer, centrifuge at 12,000 g for 30–60 seconds. Repeat once.
9. Centrifuge again at 12,000 rpm for 1 minute. Transfer the Spin column to a new tube.
10. Add 20 µL of Elution Buffer or TE, let stand for 1 minute.
11. Centrifuge at 12,000 rpm for 1 minute. The plasmid DNA is now ready for downstream use.
**Results and Discussion**
The alkaline method is one of the most commonly used techniques for plasmid isolation due to its simplicity and efficiency. It allows for the recovery of large quantities of high-quality plasmid DNA. If protein contamination occurs, the dried DNA may appear cloudy or white. Proper handling during all steps ensures the purity and integrity of the final plasmid DNA sample.
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